High performance mass spectrometry based proteomics reveals enzyme and signaling pathway regulation in neutrophils during the early stage of surgical trauma

2016 ◽  
Vol 11 (1-2) ◽  
pp. 1600001 ◽  
Author(s):  
Samina Arshid ◽  
Muhammad Tahir ◽  
Belchor Fontes ◽  
Edna Frasson de Souza Montero ◽  
Mariana S. Castro ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Signaling Pathway ◽  
High Performance ◽  
Early Stage ◽  
Surgical Trauma ◽  
Pathway Regulation

scholarly journals Front cover: High performance mass spectrometry based proteomics reveals enzyme and signaling pathway regulation in neutrophils during the early stage of surgical trauma

2017 ◽  
Vol 11 (1-2) ◽  
pp. 1770010
Author(s):  
Samina Arshid ◽  
Muhammad Tahir ◽  
Belchor Fontes ◽  
Edna Frasson de Souza Montero ◽  
Mariana S. Castro ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Signaling Pathway ◽  
High Performance ◽  
Early Stage ◽  
Surgical Trauma ◽  
Front Cover ◽  
Pathway Regulation

scholarly journals Transgelin interacts with PARP1 and affects Rho signaling pathway in human colon cancer cells

2020 ◽  
Author(s):  
Zhen-xian Lew ◽  
Hui-min Zhou ◽  
Yuan-yuan Fang ◽  
Zhen Ye ◽  
Wa Zhong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Colon Cancer ◽  
Transcription Factor ◽  
High Performance Liquid Chromatography ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
High Performance ◽  
Colon Cancer Cells ◽  
Over Expression ◽  
Key Genes

Abstract Background: Transgelin, an actin-binding protein, is associated with the cytoskeleton remodeling. Our previous studies found that transgelin was up-regulated in node-positive colorectal cancer versus in node-negative disease. Over-expression of TAGLN affected the expression of 256 downstream transcripts and increased the metastatic potential of colon cancer cells in vitro and in vivo. This study aims to explore the mechanisms that transgelin participates in the metastasis of colon cancer cells.Methods: Immunofluorescence and immunoblotting analysis were used to determine the cellular localization of the endogenous and exogenous transgelin in colon cancer cells. Co-immunoprecipitation and subsequent high performance liquid chromatography/tandem mass spectrometry were performed to identify the proteins potentially interacting with transgelin. Bioinformatics methods were used to analyze the 256 downstream transcripts regulated by transgelin to discriminate the specific key genes and signaling pathways. By analyzing the promoter region of these key genes, GCBI tools were used to predict the potential transcription factor(s) for these genes. The predicted transcription factors were matching to the proteins that have been identified to potentially interact with transgelin. The interaction between transgelin and these transcription factors was verified by co-immunoprecipitation and immunoblotting.Results: Transgelin was found to localize both in the cytoplasm and the nucleus of colon cancer cells. 297 proteins have been identified to interact with transgelin by co-immunoprecipitation and subsequent high performance liquid chromatography/mass spectrometry. Over-expression of TAGLN could lead to differential expression of 184 downstream genes. By constructing the network of gene-encoded proteins, 7 genes (CALM1, MYO1F, NCKIPSD, PLK4, RAC1, WAS and WIPF1) have been discriminated as key genes using network topology analysis. They are mostly involved in the Rho signaling pathway. Poly ADP-ribose polymerase-1 (PARP1) was predicted as the unique transcription factor for the key genes and concurrently matching to the DNA-binding proteins potentially interacting with transgelin. Immunoprecipitation validated that PARP1 interacted with transgelin in human RKO colon cancer cells.Conclusions: The results of this study suggest that transgelin binds to PARP1 and regulates the expression of the downstream key genes mainly involving Rho signaling pathway, thus participates in the metastasis of colon cancer.

scholarly journals TNF-α- Mediated-p38-Dependent Signaling Pathway Contributes to Myocyte Apoptosis in Rats Subjected to Surgical Trauma

2015 ◽  
Vol 35 (4) ◽  
pp. 1454-1466 ◽  
Author(s):  
Huaxing Wu ◽  
Guonian Wang ◽  
Shuai Li ◽  
Mingyue Zhang ◽  
Hulun Li ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
Signaling Pathway ◽  
Early Stage ◽  
Surgical Trauma ◽  
Tnf Α ◽  
Cytokine Levels ◽  
Ifn Γ ◽  
Myocyte Apoptosis ◽  
Dependent Signaling Pathway

Background: The accumulation of cytokines in the plasma after trauma can induce myocyte apoptosis. We aimed to identify which cytokine(s) present in the plasma responsible for myocyte apoptosis, and delineated the signal transduction mechanism in rats subjected to surgical trauma. Methods: Rats were randomized into two groups: control and trauma groups, which was divided into five subgroups: posttraumatic 0, 3, 6, 12, and 24 h subgroups. Cardiomyocytes isolated from traumatized rats were incubated with one of the factors for 12 h (normal plasma; Cytomix; TNF-α; IL-1β; IFN-γ; trauma plasma; anti-TNF-α antibody; SB203580). Myocyte apoptosis, cytokine levels, and MAPKs activation, as the primary experimental outcomes, were measured by TUNEL, flow cytometry, ELISA and Western blot, respectively. Results: Myocyte apoptosis was induced by surgical trauma during the early stage after trauma. Accompanying this change, plasma TNF-α, IL-1β, and IFN-γ levels were elevated in traumatized rats. Incubation of traumatized cardiomyocytes with cytomix or TNF-α alone induced myocyte apoptosis, and increased the activation of p38 and ERK1/2. Myocyte apoptosis and p38 activation were elevated in traumatized cardiomyocytes with trauma plasma, and these increases were partly abolished by anti-TNF-α antibody or SB203580. Conclusion: Our study demonstrated that there exists the TNF-α-mediated-p38-dependent signaling pathway that contributed to posttraumatic myocyte apoptosis of rats undergoing surgical trauma.

scholarly journals The Capsule Sensitizes Streptococcus pneumoniae to α-Defensins Human Neutrophil Proteins 1 to 3

2008 ◽  
Vol 76 (8) ◽  
pp. 3710-3716 ◽  
Author(s):  
Katharina Beiter ◽  
Florian Wartha ◽  
Robert Hurwitz ◽  
Staffan Normark ◽  
Arturo Zychlinsky ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Performance Liquid Chromatography ◽  
Streptococcus Pneumoniae ◽  
Human Neutrophil ◽  
High Performance ◽  
Positive Charge ◽  
Early Stage ◽  
Innate Immune ◽  
Intracellular Killing ◽  
Lipoteichoic Acids

ABSTRACT Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Its polysaccharide capsule causes resistance to phagocytosis and interferes with the innate immune system's ability to clear infections at an early stage. Nevertheless, we found that encapsulated pneumococci are sensitive to killing by a human neutrophil granule extract. We fractionated the extract by high-performance liquid chromatography and identified α-defensins by mass spectrometry as the proteins responsible for killing pneumococci. Analysis of sensitivity to the commercial α-defensins human neutrophil proteins 1 to 3 (HNP1-3) confirmed these findings. We analyzed the sensitivities of different pneumococcal strains to HNP1-3 and found that encapsulated strains are efficiently killed at physiological concentrations (7.5 μg/ml). Surprisingly, nonencapsulated, nonvirulent pneumococci were significantly less sensitive to α-defensins. The proposed mechanisms of α-defensin resistance in nonencapsulated pneumococci is surface charge modification, e.g., by introduction of positive charge by d-alanylation of surface-exposed lipoteichoic acids. These mechanisms are surmounted by the presence of the capsule, which we hypothesize is masking these charge modifications. Hence, α-defensins in the phagolysosome of neutrophils possibly contribute to intracellular killing after antibody-mediated opsonophagocytosis of encapsulated pneumococci.

scholarly journals Transgelin interacts with PARP1 and affects Rho signaling pathway in human colon cancer cells

2020 ◽  
Author(s):  
Zhen-xian Lew ◽  
Hui-min Zhou ◽  
Yuan-yuan Fang ◽  
Zhen Ye ◽  
Wa Zhong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Colon Cancer ◽  
Transcription Factor ◽  
High Performance Liquid Chromatography ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
High Performance ◽  
Colon Cancer Cells ◽  
Over Expression ◽  
Key Genes

Abstract Background: Transgelin, an actin-binding protein, is associated with the cytoskeleton remodeling. Our previous studies found that transgelin was up-regulated in node-positive colorectal cancer versus in node-negative disease. Over-expression of TAGLN affected the expression of 256 downstream transcripts and increased the metastatic potential of colon cancer cells in vitro and in vivo. This study aims to explore the mechanisms that transgelin participates in the metastasis of colon cancer cells.Methods: Immunofluorescence and immunoblotting analysis were used to determine the cellular localization of the endogenous and exogenous transgelin in colon cancer cells. Co-immunoprecipitation and subsequent high performance liquid chromatography/tandem mass spectrometry were performed to identify the proteins potentially interacting with transgelin. Bioinformatics methods were used to analyze the 256 downstream transcripts regulated by transgelin to discriminate the specific key genes and signaling pathways. GCBI tools were used to predict the potential transcription factor(s) for these key genes. The predicted transcription factors were matching to the proteins that have been identified to interact with transgelin. The interaction between transgelin and these transcription factors was verified by co-immunoprecipitation and immunofluorescence.Results: Transgelin was found to localize both in cytoplasm and nucleus of colon cancer cells. 297 proteins have been identified to interact with transgelin by co-immunoprecipitation and subsequent high performance liquid chromatography/mass spectrometry. Over-expression of TAGLN could lead to differential expression of 184 downstream genes. The network of gene-encoded proteins was constructed in which 7 genes (CALM1, MYO1F, NCKIPSD, PLK4, RAC1, WAS and WIPF1) have been discriminated as key genes by network topology analysis. They are mostly involved in the Rho signaling pathway. Poly ADP-ribose polymerase-1 (PARP1) was predicted as the unique transcription factor for the key genes and concurrently matching to the DNA-binding proteins potentially interacting with transgelin. Immunoprecipitation and immunofluorescence validated the interaction between PARP1and transgelin in human RKO colon cancer cells. Conclusions: The results of this study suggest that transgelin binds to PARP1 and regulates the expression of downstream key genes which are mainly involved in Rho signaling pathway, thus participates in the metastasis of colon cancer.

scholarly journals Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability

2015 ◽  
Vol 21 (2) ◽  
pp. 156-164 ◽  
Author(s):  
Laurie J. Gordon ◽  
Morven Allen ◽  
Per Artursson ◽  
Michael M. Hann ◽  
Bill J. Leavens ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Direct Measurement ◽  
High Performance ◽  
Early Stage ◽  
Cell Permeability ◽  
Tandem Mass ◽  
Cellular Activity ◽  
Production Scale ◽  
Compound Concentration

One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.

scholarly journals Transgelin interacts with PARP1 and affects Rho signaling pathway in human colon cancer cells

2020 ◽  
Author(s):  
Zhen-xian Lew ◽  
Hui-min Zhou ◽  
Yuan-yuan Fang ◽  
Zhen Ye ◽  
Wa Zhong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Colon Cancer ◽  
Transcription Factor ◽  
High Performance Liquid Chromatography ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
High Performance ◽  
Colon Cancer Cells ◽  
Over Expression ◽  
Key Genes

Abstract Background: Transgelin, an actin-binding protein, is associated with the cytoskeleton remodeling. Our previous studies found that transgelin was up-regulated in node-positive colorectal cancer versus in node-negative disease. Over-expression of TAGLN affected the expression of 256 downstream transcripts and increased the metastatic potential of colon cancer cells in vitro and in vivo. This study aims to explore the mechanisms that transgelin participates in the metastasis of colon cancer cells. Methods: Immunofluorescence and immunoblotting analysis were used to determine the cellular localization of the endogenous and exogenous transgelin in colon cancer cells. Co-immunoprecipitation and subsequent high performance liquid chromatography/tandem mass spectrometry were performed to identify the proteins potentially interacting with transgelin. Bioinformatics methods were used to analyze the 256 downstream transcripts regulated by transgelin to discriminate the specific key genes and signaling pathways. By analyzing the promoter region of these key genes, GCBI tools were used to predict the potential transcription factor(s) for these genes. The predicted transcription factors were matching to the proteins that have been identified to potentially interact with transgelin. The interaction between transgelin and these transcription factors was verified by co-immunoprecipitation and immunoblotting. Results: Transgelin was found to localize both in the cytoplasm and the nucleus of colon cancer cells. 297 proteins have been identified to interact with transgelin by co-immunoprecipitation and subsequent high performance liquid chromatography/mass spectrometry. Over-expression of TAGLN could lead to differential expression of 184 downstream genes. By constructing the network of gene-encoded proteins, 7 genes ( CALM1, MYO1F, NCKIPSD, PLK4, RAC1, WAS and WIPF1 ) have been discriminated as key genes using network topology analysis. They are mostly involved in the Rho signaling pathway. Poly ADP-ribose polymerase-1 (PARP1) was predicted as the unique transcription factor for the key genes and concurrently matching to the DNA-binding proteins potentially interacting with transgelin. Immunoprecipitation validated that PARP1 interacted with transgelin in human RKO colon cancer cells. Conclusions: The results of this study suggest that transgelin binds to PARP1 and regulates the expression of the downstream key genes mainly involving Rho signaling pathway, thus participates in the metastasis of colon cancer.

888: Metabolomic Urine Samples Profiling Using High Performance Liquid Chromatography Coupled with Mass Spectrometry to Detect Bladder Cancer

2007 ◽  
Vol 177 (4S) ◽  
pp. 295-295
Author(s):  
Michael Mullerad ◽  
Haleem J. Issaq ◽  
Alexander Kravtsov ◽  
Timothy Waybright ◽  
Brian Luke ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bladder Cancer ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Urine Samples
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